Synthesis of steroids with kabatiella phoradendri



SYNTHESIS OF STEROIDS WITH KABATIELLA PHORADENDRI Allen I. Laskin,Franklin Township, Mariano A. Guiducci, Edison, and Josef Fried,Princeton, N.J.,,assignors to Olin Mathieson Chemical Corporation, NewYork, N.Y., a corporation of Virginia No Drawing. Filed May 22, 1959,Ser. No. 814,986 Claims. (Cl. 195--51) This invention relates to, andhas for its object the provision of, a method of producing ZI-hydroxysteroids of the pregnane series. These steroids are known compoundswhich are either physiologically active or are useful in the preparationof physiologically active steroids by methods known in the art.

It has been found that steroids of the pregnane series (especially ofthe 3,20-diketo-A -pregnene series), unsubstituted in the Zl-position,can be converted to their corresponding ZI-hydroxy derivatives bysubjecting the former to the action of enzymes of the known species ofmicroorganism, Kabatiella phoradendri. The action of the enzymes can beutilized either by bringing together, in an aqueous nutrient medium, thesteroid, oxygen and enzymes or non-proliferating cells of themicroorganism or (preferably) by including the steroid in an aeratedculture of the microorganism.

In general, the conditions of culturing the microorganism for thepurpose of this invention are (except for the inclusion of the steroidto be 2l-hydroxylated) the same as those of culturing microorganisms forthe production of antibiotics or vitamins; thus, the microorganism isgrown in contact with (in or on) a suitable nutrient medium in thepresence of oxygen (air). A suitable nutrient medium essentiallycomprises a source of nitrogenous factors and an assimilable source ofcarbon and energy. The latter may be a carbohydrate (such as sucross,molasses, glucose, maltose, starch, or dextrin) and/ or the steroiditself. Preferably, however, the medium includes an assimilable sourceof carbon and energy in addition to the steroid.

The source of nitrogenous factors may be organic (e. g. soybean meal,corn' steep liquor, meat extract, distillers solubles, peptones and/oryeast extract) or synthetic (i. e. composed of simply, synthesizableorganic and in organic compounds such as ammonium salts, alkalinitrates, amino acids or urea).

Among the 21-unsubstituted steroids of the pregnane series which may beconverted into useful ZI-hydroxy derivatives by the practice of thisinvention may be mentioned progesterone; A -dehydroprogesterone;pregnenolone; pregnane-3,20-dione; monohydroxyprogesterones [e. g.,1la-hydroxyprogesterone, the 9a.- and 12u-halollfi-hydroxyprogesterones,17e-hydroxyprogesterone, and the 9ccand IZOC-IOWeI' alkyl (e. g.II'IBlhYD-IIIS-hYdIOXY- progesterones]; the dihydroxyprogesterones [e.g.1113, 170a dihydroxyprogesterone, A pregnadiene 1l 3,17ediol-3,20-dione,the 9m and IZa-halo-l1,8,17e-dihydroxyprogesterones, the 90o andl2a-halo-A -pregnadiene-11,8, 17OL-diOl-3,20-dlOIiCS, and the 9:1- andl2u-lower alkyl (e. g. methyl)-l1B,17u-dihydroxyprogesterones]; thetrihydroxyprogesterones [e g. l1 3,16a,l7a-trihydroxyprogesterone, A-pregnadiene-llp,16a,l7a-triol-3, ZO-dione, the 911- andIZu-halo-l1B,l6a,l7oc-trihydroxyprogesterones, the 9aand 12a-halo-A-pregnadiene-l1B-16u,17atriol-3, 20-diones, and the 9aand 12a-loweralkyl (e.g.

States Patent 0 methyl)-11e,16a,17a-trihydroxyprogesterones]; and thell-keto analogues of those steroids containing an 113- hydroxy group. I1

The following examples are illustrative of the invention (alltemperatures being in centrigrade):

EXAMPLE 1 1 Za-meIhyI-I 1 -dehydr0c0rticosterone '(a) Fermentation:Surface growth of a two week old agar slant, culture of Kabatiellaphoradendri (American Type Culture Collection No. 11,129), the slantcontaining as a nutrient medium (A) Glucose 10 Yeast extract 2.5

K HPO 1 Agar 20 Distilled water to 1 liter.

is suspended in 2.5 ml. of a 0.01% Dupanol aqeous solution. One ml.portions of the suspension are used to inoculate ten 250 ml. conicalflasks, each containing 50 ml. of the following sterilized medium (B):

G. Dextrose 10 Corn steep liquor 6 NH H PO 3 Yeast extract 2.5 CaCO 2.5Soybean oil 2.2

Distilled water to 1 liter.

tents of the flasks are pooled and filtered through a Seitz,

clarifying pad. The flasks, mycelium and pads are washed with successive50 ml. portions of warm water. The combined filtrate and washings have avolume of 3350 m1.

(b) isolation of 12a-methyl-l l-dehydrocorticosterone: The combinedfiltrate and washings are extracted with two 500 ml. and two 400 ml.portions of chloroform. The combined chloroform extracts are then washedsuccessively with 1% hydrochloric acid, water, sodium bicarbonatesolution and again with water, dried over sodium sulfate, filtered andevaporated to dryness in vacuo. The resulting crude product weighs about740 mg. Crystallization from ethanol yields about 513 mg. of-material(49% yield) M.P. about -149. The analytical sample has the followingproperties: M.P. about 149-151"; [ot] +2O7.5 (CHCl N22; 237 s fiti'."5.90, 6.05 and 6.19

Analysis.--Calcd for C H O (358): C, 73.71; H, 8.44. Found: C, 73.54; H,8.47.

2.78, 2.92, 5.86 (inflection),

EXAMPLE 2 1 1 -desoxycorticosterone EXAMPLE 3 A-pregnadiene-21-0l-3,20-di0ne Following the procedure of Example 1, butsubstituting 5 an equivalent amount of l6-dehydroprog esterone for theIZa-methyI-Il-ketoprogesterone in step a, M -pregnadiene-21-ol-3,20-dione is obtained.

The invention may be variously otherwise embodied within the scope ofthe appended claims.

What is claimed is:

1. A process for preparing a ll-hydroxylated steroid of the pregnaneseries, which comprises subjecting a steroid of the pregnane series,unsubstituted in the 21- position, to the action of enzymes of themicroorganism 4 Kabatiella phoradendri in the presence of oxygen andrecovering the resulting 21-hydroxy steroid.

2. The process of claim 1 wherein the steroid reactant is of the3,20-diketo-A -pregnene series.

3. The process of claim 1 wherein the steroid reactant is progesterone.

4. The process of claim 1 wherein the steroid reactant is A-dehydroprogesterone.

5. The process of claim 1 wherein the steroid reactant 10 is12oz-methyl1l-ketoprogesterone.

References Cited in the file of this patent UNITED STATES PATENTSHagemann et al Sept. 10, 1957

1. A PROCESS FOR PREPARING A 21-HYDROXYLATED STERIOD OF THE PREGNANESERIES, WHICH COMPRIISES SUBJECTING A STEROID OF THE PREGNANE SERIES,UNSUBSTITUTED IN THE 21POSITION, TO THE ACTION OF ENZYMERS OF THEMICROORGANISM KABATIELLA PHORADENDRI IN THE PRESENCE OF OXYGEN ANDRECOVERING THE RESULTING 21-HYDROXY STEROID.